Human Immunodeficiency Virus-Type 1 (HIV-1) is the etiologic agent that is responsible for Acquired Immunodeficiency Syndrome (AIDS), a syndrome characterized by depletion of CD4+ T-lymphocytes and collapse of the immune system. HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. At present, the number of persons infected with the pathogenic virus, HIV, has exceeded 33,000,000 all over the world, and about 2,500,000 persons are being newly infected every year.
Helper CD4 T-cells are the most important cells in maintaining the body's powerful immunity and they are required for almost all our immune responses. As dramatically demonstrated in acquired immunodeficiency syndrome (AIDS) patients, a person lacking CD4 T-cells cannot fend off even microbes that are normally harmless. AIDS is caused by the human immunodeficiency virus (HIV), which infects and kills CD4 T-cells.
When the disease progresses from HIV-1 infection to full-blown AIDS, it is because the number of T-cells has dropped to dangerous levels. AIDS is heralded by a total lymphocyte count of less than 500/mm3 and a dangerously low T-cell count of below 200/mm3. With the immune system so depleted, the body becomes highly vulnerable to opportunistic diseases. As the term suggests, these are infections and other diseases that seize the opportunity presented by a weakened defense system. They commonly include herpes simplex infection and other herpes conditions such as shingles and the oral yeast infection, thrush; Kaposi's sarcoma, characterized by the dark lesions; CKV retinitis, a herpes virus that can bring blindness; meningitis, an infection of the spinal cord and brain; cervical cancer; tuberculosis, and a formerly rare type of pneumonia.
Despite extensive efforts over the past quarter century, the precise mechanism by which HIV-1 causes progressive depletion of CD4 T-cells remains debated. Both direct and indirect cytopathic effects have been proposed. When immortalized T-cell lines are infected with laboratory-adapted HIV-1 strains, direct CD4 T-cell killing predominates. Conversely, in more physiological systems, such as infection of lymphoid tissue with primary HIV-1 isolates, the majority of dying cells appear as uninfected “bystander” CD4 T-cells (Finkel et al., 1995, Nat Med 1:129-134; Jekle et al., 2003, J Virol 77:5846-5854).
Various mechanisms have been proposed to contribute to the death of these bystander CD4 T-cells including the action of host-derived factors like tumor necrosis factor-α, Fas ligand and TRAIL (Gandhi et al., 1998, J Exp Med 187:1113-1122; Herbeuval et al., 2005, Blood 106:3524-3531), and viral factors like HIV-1 Tat, Vpr, and Nef released from infected cells (Schindler et al., 2006, Cell 125:1055-1067; Westendorp et al., 1995, Nature 375:497-500). Considerable interest has also focused on the role of gp120 and gp41 Env protein in indirect cell death, although it is not clear whether death signaling involves gp120 binding to its chemokine receptor or gp41-mediated fusion. It is also unclear whether such killing is caused by HIV-1 virions or by infected cells expressing Env.
Most studies have focused on death mechanisms acting prior to viral entry. Less is known about the fate of HIV-1-infected CD4 T-cells that do not express viral genes, in particular naive CD4 T-cells in tissues that are refractory to productive HIV infection (Glushakova et al., 1995, Nat Med 1:1320-1322; Kreisberg et al., 2006, J Exp Med 203:865-870). In these cells, infection is aborted after viral entry, as reverse transcription is initiated but fails to reach completion (Kamata et al., 2009, PLoS Pathog 5, e1000342; Epub 129 March 1000320; Swiggard et al., 2004, AIDS Res Hum Retroviruses 20:285-295; Zack et al., 1990, Cell 61:213-222; Zhou et al., 2005, J Virol 79:2199-2210).
Human lymphoid aggregate cultures (HLACs) prepared from tonsillar tissue closely replicate the conditions encountered by HIV in vivo and thus form an attractive, biologically relevant system for studying HIV-1 infection (Eckstein et al., 2001, Immunity 15:671-682). Lymphoid organs are the primary sites of HIV replication and contain more than 98% of the body's CD4 T-cells. Moreover, events critical to HIV disease progression occur in lymphoid tissues, where the network of cell-cell interactions mediating the immune response deteriorates and ultimately collapses. Primary cultures of peripheral blood cells do not fully mimic the cytokine milieu, the cellular composition of lymphoid tissue, nor the functional relationships that are undoubtedly important in HIV pathogenesis. Finally, HLACs can be infected with a low number of viral particles in the absence of artificial mitogens, allowing analysis of HIV cytopathicity in a natural and preserved environment.
In studies described more fully herein (e.g., see, Examples), it was discovered that the death of so-called uninfected “bystander” T-cells involves abortive HIV-1 infection. More specifically, it was discovered that after viral entry, incomplete HIV-1 reverse transcriptase products activate a host defense program that elicits a coordinated proapoptotic and proinflammatory response involving activation of the enzymes caspase-1 and caspase-3.
Caspases are a family of at least fourteen cysteine-dependent aspartate-directed proteases that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, 1998, Chem Biol 5:R97-R103). These signaling pathways vary depending on cell type and stimulus, but all apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.
Caspase-1, the first identified caspase, is also known as interleukin converting enzyme or “ICE.” Caspase-1 exists as an inactive proenzyme, which undergoes proteolytic processing at conserved aspartic residues to produce two subunits, large (caspase-1 p20 subunit) and small, (caspase-1 p10 subunit) that dimerize to form the active enzyme. Caspase-1 polypeptides are derived from various caspase-1 isoform precursors. Caspase-1 converts the inactive precursor of interleukin-1-beta (pIL-1β) to the pro-inflammatory active form by specific cleavage of pIL-1β between Asp-116 and Ala-117. Besides caspase-1 there are also eleven other known human caspases, all of which cleave specifically at aspartyl residues. They are also observed to have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The group of caspases, which includes caspases 1, 4, 5 and 11, have been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site (preferred sequence Trp-Glu-His-Asp (SEQ ID NO: 1)). Another group, which includes caspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10, tolerate many amino acids in the primary recognition sequence, but seem to prefer residues with branched, aliphatic side chains such as valine and leucine at position 1 (Leu/Val-Glu-X-Asp) (SEQ ID NO: 2).
The caspases have also been grouped according to their perceived function. The first subfamily consists of caspases-1 (ICE), 4, 5, 11 and 12. These caspases have been shown to be involved in pro-inflammatory cytokine processing and therefore play an important role in inflammation. Caspase-1, the most studied enzyme of this class, activates the IL-1β precursor by proteolytic cleavage. This enzyme therefore plays a key role in the inflammatory response. Applicants, however, are unaware of anything in the art suggesting the use of caspase-1 inhibitors in methods for the treatment of an HIV-1 infection and AIDS and for use in related methods described herein.
The remaining caspases make up the second and third subfamilies. These enzymes are of central importance in the intracellular signaling pathways leading to apoptosis. One subfamily consists of the enzymes involved in initiating events in the apoptotic pathway, including transduction of signals from the plasma membrane. Members of this subfamily include caspases-2, 8, 9 and 10. The other subfamily, consisting of the effector caspases 3, 6 and 7, are involved in the final downstream cleavage events that result in the systematic breakdown and death of the cell by apoptosis. Caspases involved in the upstream signal transduction activate the downstream caspases, which then disable the DNA repair mechanisms, fragment the nuclear DNA, dismantle the cell cytoskeleton and finally fragment the cell.
Caspase-3 (also known as apopain, CPP-32, and YAMA) is responsible for proteolytic cleavage of a variety of fundamental proteins, including cytoskeletal proteins, kinases and DNA-repair enzymes. It is a critical mediator of apoptosis in neurons. Caspase-3 exists as an inactive proenzyme, which undergoes proteolytic processing at conserved aspartic residues to produce two subunits, large (caspase-3 p17 subunit) and small, (caspase-3 p12 subunit) that dimerize to form the active enzyme. Caspase-3 polypeptides are derived from various caspase-3 isoform precursors. Inhibition of caspase-3 has shown efficacy in models, such as stroke, traumatic brain spinal cord injury, hypoxic brain damage, cardiac ischemia and reperfusion injury. Inhibition of caspase-1 has been shown to be beneficial in models of, e.g., rheumatoid arthritis, osteoarthritis, inflammatory bowel disease and asthma. However, as much as Applicants are aware, hitherto, nothing in the art suggested the use of caspase-1 or caspase-3 inhibitors in methods for the treatment of an HIV-1 infection and AIDS and for use in related methods described herein.
The present treatments available for HIV-1 infection and AIDS seek to block one or more steps involved in the production of viral particles and often are based on a combination of several drugs, a so-called cocktail of inhibitors of reverse transcriptase and protease inhibitors. Treatment options involve administration of reverse transcriptase inhibitors, inhibitors of viral protease, fusion, entry, or integration inhibitors in different combinations to block multiple steps in the viral life cycle. This approach, termed highly active antiviral therapy (HAART) has greatly decreased morbidity and mortality in people infected with HIV (Palella et al., 1998, N Engl J Med 338(13):855-860). While HAART is quite effective and can reduce the virus back to undetectable levels in patient's blood, it is not a cure for the patient, because the virus is still present in the immune cells, and the disease can reappear at any time due to emergence of drug-resistant viruses; upon discontinuation of therapy viremia peaks and rapid progression to AIDS is frequently observed. Furthermore, the immunodeficiency and the HIV-1 specific T-cell dysfunction persist during HAART. This therapy requires life-long treatment and the treatment is very expensive. The cost of the drugs alone often exceeds USD 15,000. There are, in addition, several other problems associated with this therapy; difficulties with patient compliance (complicated drug regimens), development of resistant viruses, non-ideal pharmacokinetics and side effects such as, for example, suppression of bone-marrow and long-term metabolic effects.
The global health crisis caused by HIV-1 is unquestioned, and while recent advances in drug therapies have been successful in slowing the progression of AIDS, there is still a need to find a safer, more efficient, less expensive way to control the virus, to treat patients having an HIV-1 infection or AIDS. Applicants herein provide novel methods for the treatment of HIV-1 infection and AIDS that overcome the afore-mentioned problems.